Inflammatory bowel disease (IBD) is a kind of chronic inflammatory disease with its etiology and pathogenesis being unclear up to now. At present, the common clinical IBD includes Crohn's disease (CD) and Ulcerative colitis (UC, also known as chronic non-specific UC), which are reported as high incidence rate, long duration, and recurrent attacks and the like. With increasing knowledge for this disease in the field of medical sciences and the development of medical diagnostic tools in recent years, clinical statistics has made it very clear that approximately 5%-7% of patients of UC will progress toward malignant transformation and probably will lead to cancerization ultimately arisen from UC, with serious dysplasia of intestinal glands and carcinoma of colon and rectum often being formed, and pathologically, the incidence of undifferentiated-type predominates in patients of UC, and too often with high degree of deterioration and poor prognosis. At present, the lack of efficient drug and other effective therapy for the treatment of UC is severe, leading to no complete cure to UC clinically. So it has been identified as nasty disease in the field of medical sciences, which seriously affected the lives of the patients. (At present, there are only several drugs, such as mesalazine (e.g. SASP etc.), immunosuppressive agents, and hormone and on the like, being used to treat UC clinically. Although some efficacy is observed, many problems abound nowadays, such as relapse after treatment and serious side effect, among others).
The lesions of UC are mainly confined to the mucosal layer of colon, too often with ulcer being the most dominant symptom and relapse after treatment and severe gastrointestinal inflammation being its characteristics, which also often implicates rectum and distal colon. These symptoms can also extend to the proximal end, and even the entire colon. According to national collaborative group of IBD, the incidence rate of UC was about 11.62 cases in 100,000 people each year in China, and hospitalized UC patients were mainly mild (35.4%) and moderate (42.9%) cases. In western countries, the morbidity of UC was 79-268 cases in 100,000 people each year, while, in Asia in the late 20th century, it was 7.8-18.1 cases and 8.6 cases in 100,000 people each year in Japan and Singapore, respectively. In recent years, morbidity of UC has shown a tendency of increasing across China (Maybe it is due to our increasing understanding for this disease and the development of medical diagnostic tools). Realistically, getting UC would make patients suffer not only from mental and physical pain, but also from heavy economic burden to themselves.
At the early stage of UC, there can be diffuse inflammation of mucosa, edema, hyperemia, and local hemorrhage, and with diffuse fine granule in the mucosal surface, fragile organization, and easy bleeding when touched also often being observed. Lymphocytes, plasma cells, eosinophils and neutrophils infiltration can be found in the mucosa and submucosa. With the progression of the disease, a large number of neutrophils will gather in the bottom of intestinal glands, leading to the formation of a small crypt abscess. When the crypt abscess are mixed together and broken up, mucosa will show wide shallow small irregular ulcer. The ulcer may develop along the longitudinal axis of colon, merging into irregular large ulcer gradually. In the process of repeated episodes of chronic colitis, a lot of new granulation tissue will proliferate, often together with inflammatory polyp. Due to the continuous damage and repair, mucosa will lose its normal structure and fibrous tissue will increase, leading to atrophic changes such as glands being degenerated, arranged in disorder, and reduced in number. With the healing of ulcer, forming of scar, and hypertrophying of muscular layer of mucosa and muscle layer, the colon will deform, colonic pouch will disappear, and even intestinal lumen will narrow, leading to the organic and functional changes of colon, which will seriously affect the health of human body.
Recent investigations have shown that the occurrence and progression of chronic IBD, including CD and UC, are closely related to an imbalance of microenvironment homeostasis function of intestinal epithelial cells. The disorder of homeostasis function of intestinal epithelial cell can trigger non-controllable endoplasmic reticulum stress response (unresolving ER stress), leading to extensive and persistent endoplasmic reticulum damage in intestinal epithelial cells, often with “programmed cell death”, namely, apoptosis. Also, it has been reported that the occurrence and progression of IBD are closely related to the non-controllable endoplasmic reticulum stress response within intestinal epithelial cells. Especially the abnormality of key downstream transcription factor, X-box-binding protein 1 (xbp1), which associates with non-controllable endoplasmic reticulum stress response within intestinal epithelial cells plays a key role in the incidence of UC.
Generally, the transcription factor xbp1 plays a very important role for the expansion of endoplasmic reticulum and the growth of glandular epithelial cells sharing secretion function, such as plasma cells, islet cells in the pancreas, and salivary gland cells, and for the adaption of epithelial cells to inflammatory stimulation environment. Of almost all cell types from human body, xbp1 serve as a key regulator in maintaining the basic function of endoplasmic reticulum through directly regulating the transcriptional function of a core set of genes. Kaser and coauthors have created a gene knockout mouse model with xbp1 defect of intestinal epithelial cells (XBP1−/−) in 2008 for the first time. They found that animals with xbp1 gene knockout would generate not only the deficiency of Paneth cell and the obvious change of phenotype of goblet cell, but also the spontaneous inflammatory changes in the ileum. At the same time, further discovery was made by researchers on the basis of the above findings that the sense mutant of the xbp1 gene also related closely to the occurrence and progression of IBD. The above experimental results demonstrated that xbp1 gene plays an important role in maintaining the homeostasis of intestinal epithelial cells and in resisting the apoptosis of intestinal epithelial cell induced by ER stress. Similarly, by taking the SNPs technology in clinical studies, it was found that patients getting IBD typically show some variations in the coding region of the xbp1 gene, making them to be more sensitive to predisposing factors of IBD. Taken together, according to research data from not only clinical genetics but also in laboratories, it can be make clear that transcription factor xbp1 plays a very important role in the self homeostasis regulation of intestinal epithelial cell. The failure or weakness of xbp1 expression will increase the sensibility of body to inducing factor of IBD, promote getting IBD, and lead to deterioration of IBD.
However, there has no claim as yet to chemically synthesized small molecule drug or natural medicine monomer in the field of developing innovative anti-IBD drugs with xbp1 as target during recent research across the world. Base on the above findings from laboratory to clinical test, i.e., the research information about the close relationship between the expressive failure and abnormality of xbp1 and the increasing morbidity of IBD, it is speculated that xbp1 may be the potential new drug target for treating IBD. Therefore, the object of the present invention is to screen and discovery selective agonists of xbp1 on the basis of different aspects such as gene transcription regulation of xbp1, mRNA expression, and protein synthesis and the like by establishing drug screening model in vitro with xbp1 as target, in combination with cytology and molecular biology means, mainly including dual luciferase reporter gene, real-time quantitative polymerase chain reaction (PCR) and Western Blot (WB) techniques.
Moreover, the present invention also provides a reliable, accurate, and effective method for discovering anti-IBD drug by high-throughput screening.
The present invention obtains some protoberberine alkaloid derivatives or their physiologically acceptable salts by structure modifications of kinds of protoberberine alkaloid quaternium. In pharmacodynamic experiments at molecular and animal level, these protoberberine alkaloid derivatives show certain or significant anti-UC activity with a few of them showing far more efficiency than substrates and positive drug. Especially, the above mentioned compounds 1, 2, and 7 show significant transcriptional activation effect on xbp1 gene at molecular level in vitro, wherein the EC50 values are 2.29×10−9 (mol/L), 7.06×10−9 (mol/L), and 2.21×10−7 (mol/L), respectively. On the other hand, in vivo experiments show that the disease activity index (DAI) (including mental state, weight loss, bloody stool, shape of stool and other evaluation indicators) inhibitory rate of compound 7 (500 mg/kg) in UC model is up to 64%, and on the case of compound 1 (300 mg/kg) and 2 (300 mg/kg), the inhibition rates are as high as 69% and 80%, respectively, while the positive drug SASP (300 mg/kg) is only 32%. In addition, the histopathological test results show that the high-dose group of compound 7 (500 mg/kg) has significant improvement on the colon inflammatory lesion, with intestinal epithelial cells arranged perfectly, and even the cell polarity arrangement can recover to the normal physiological state. Therefore, the results from in vivo experiments of different animal species and different pathogenesis demonstrate that the protoberberine alkaloid derivatives obtained in the present invention exhibit far more significant anti-UC activity in vivo than those currently used clinically, such as SASP, and thus they have important medicinal value in preparing drugs for the treatment of UC. In addition, comparing with substrates, the solubility of these prepared protoberberine alkaloid derivatives has also been significantly improved, especially in those poor solvents for substrates.